Summary HIV-associated neurocognitive disorders (HANDs) and substance abuse comorbidity remain prevalent despite combination antiretroviral therapy (cART). The innovative hypothesis behind this project is that reversing trophic deficits that characterize HANDs and manifest as synaptodendritic and mitochondrial injury can effectively prevent or ameliorate cognitive decline in HIV and compulsive drug taking. To test this hypothesis, we will perform a throughput screening campaign (HTS) to identify modulators of a newly identified gene regulatory mechanism that broadly regulates trophic support and mitochondrial biogenesis in key cortical and hippocampal neuronal populations and that we found are downregulated in both neuroAIDS and dependence. We expect that small molecules that are able to upregulate this mechanism will make neurons more resilient to oxidative stress and neurodegeneration, resulting in a reduction of neuronal injury and hypofrontality, which is a key cause of cognitive dysfunction in HIV and compulsive drug and alcohol taking in dependent individuals. We will employ cell-based luminescence reporter assays in a 1536-well plate format in conjunction with a tiered approach to screen the >640,000-compound Scripps Drug Discovery Library (SDDL) and confirm the potency of ~500 drug-like molecules. Hit-validation will be performed to identify small-molecule regulators and eliminate nonspecific effectors using parallel orthogonal assays and off-target assessment using cytotoxicity counterscreens employing disease-relevant cell types. To prioritize hit scaffold series, we will select analogs of confirmed hits from both the existing compound libraries and other commercial sources. Hit scaffolds will be triaged to remove intractable molecules. We will select 3-5 molecular series from the most promising hits which, will be profiled to verify the selectivity, potency, and lack of cytotoxicity. Using an orchestrated effort from the applicant laboratories (Scripps California and Scripps Florida), leads in 2-4 series will be formulated and retested for potency/selectivity with the aim of advancing leads that can elicit the appropriate in vitro response in the aforementioned assays and can be evaluated for appropriate phenotypic responses to cell-expression levels. This will be followed by in vitro and in vivo pharmacokinetics (PK) studies to identify 1-2 top scaffolds for further investigation. Finally, the most promising 2-3 compounds with favorable drug metabolism and pharmacokinetics (DMPK) properties will be selected for in vivo testing, including high oral bioavailability, and will be scaled up and tested in vivo in rodent efficacy models. Efficacy in regulating the expression of the gene expression program under study will be validated by quantitative polymerase chain reaction of synaptic, dendritic, and mitochondrial genes and RNA-Seq in conjunction with Gene Set Enrichment Analyses (GSEA). We will then test the ability of the optimized probe compounds to prevent or ameliorate cognitive impairment that is attributable to prefrontal and hippocampal degenerative changes in HIV-1 transgenic rats with and without concomitant compulsive drug or alcohol self-administration.